Patient oxygenation using stabilized fluorocarbon emulsions

ABSTRACT

Storage stable fluorocarbon emulsions having a continuous aqueous phase and a discontinuous fluorocarbon phase, in which the fluorocarbon phase comprises a major amount of a first fluorocarbon or fluorocarbon mixture, and a minor amount of a second fluorocarbon or fluorocarbon mixture, in which the second fluorocarbon has a molecular weight greater than that of the first fluorocarbon and the second fluorocarbon includes a lipophilic moiety in its structure, whereby the second fluorocarbon serves to promote particle size stability in the emulsion while simultaneously providing favorably short organ retention times when administered to animals in vivo.

RELATED APPLICATIONS

This application is a continuation of prior U.S. patent application Ser.No. 10/007,053, filed Dec. 3, 2001, which is a continuation of priorU.S. patent application Ser. No. 09/263,924, filed Mar. 5, 1999, whichis a continuation of U.S. patent application Ser. No. 08/854,547, filedMay 12, 1997, which is a continuation of U.S. patent application Ser.No. 07/967,700, filed Oct. 27, 1992, now U.S. Pat. No. 5,628,930.

BACKGROUND OF THE INVENTION

The present invention relates to emulsions comprising highly fluorinatedor perfluorinated compounds. More particularly, it relates tofluorocarbon emulsions having superior particle size stability duringstorage.

Fluorocarbon emulsions find uses as therapeutic and diagnostic agents.Most therapeutic uses of fluorocarbons are related to the remarkableoxygen-carrying capacity of these compounds. One commercial biomedicalfluorocarbon emulsion, Fluosol (Green Cross Corp., Osaka, Japan), ispresently used as a gas carrier to oxygenate the myocardium duringpercutaneous transluminal coronary angioplasty (R. Naito, K. Yokoyama,Technical Information Series No. 5 and 7, 1981). Fluorocarbon emulsionshave also been used in diagnostic applications such as imaging.Radiopaque fluorocarbons such as perflubron (perfluorooctyl bromide orC₈F₁₇Br) are particularly useful for this purpose.

It is important that fluorocarbon emulsions intended for medical useexhibit particle size stability. Emulsions lacking substantial particlesize stability are not suitable for long term storage, or they requirestorage in the frozen state. Emulsions with a short shelf life areundesirable. Storage of frozen emulsions is inconvenient. Further,frozen emulsions must be carefully thawed, reconstituted by admixingseveral preparations, then warmed prior to use, which is alsoinconvenient.

Davis et al., U.S. Pat. No. 4,859,363, disclose stabilization ofperfluorodecalin emulsion compositions by mixing a minor amount of ahigher boiling perfluorocarbon with the perfluorodecalin. Preferredhigher boiling fluorocarbons were perfluorinated saturated polycycliccompounds, such as perfluoroperhydrofluoranthene. Others have alsoutilized minor amounts of higher boiling fluorocarbons to stabilizeemulsions. See, e.g., Meinert, U.S. Pat. No. 5,120,731 (fluorinatedmorpholine and piperidine derivatives), and Kabalnov, et al., KolloidnZh. 48: 27-32 (1986)(F—N-methylcyclohexylpiperidine).

Davis, et al. suggested that the primary phenomenon responsible forinstability of small particle size fluorocarbon emulsions was Ostwaldripening. During Ostwald ripening, an emulsion coarsens throughmigration of molecules of the discontinuous phase from smaller to largerdroplets. See generally, Kabalnov, et al., Adv. Colloid Interface Sci.38: 62-97 (1992). The force driving Ostwald ripening appears to berelated to differences in vapor pressures that exist between separatedroplets. Such a difference in vapor pressure arises because smallerdroplets have higher vapor pressures than do larger droplets. However,Ostwald ripening may only proceed where the perfluorocarbon moleculesare capable of migrating through the continuous phase between dropletsof the discontinuous phase. The Lifshits-Slezov equation relates Ostwaldripening directly to water solubility of the discontinuous phase. SeeLifshits, et al., Sov. Phys. JETP 35: 331 (1959).

It is known that addition of higher molecular weight compounds, havinglower vapor pressures and lower solubility in the continuous phase,reduces such interparticle migration. This, in turn, reduces Ostwaldripening and improves particle size stability. Thus, the conventionalprior art solution to the particle size stability problem is to add acertain amount (e.g., 10-30% of the fluorocarbon content) of a highermolecular weight fluorocarbon to the discontinuous phase.

Fluorocarbon emulsion particles are taken up and temporarily retained bycells of the reticuloendothelial system (RES). It is desirable tominimize this retention time. Unfortunately, when the prior art includedhigher molecular weight fluorocarbons in fluorocarbon emulsions, organretention times were also increased considerably. Organ retention timefor most fluorocarbons bears an exponential relationship to themolecular weight of the fluorocarbon. See J. G. Riess, Artificial Organs8: 44, 49-51; J. G. Riess, International Symposium on Blood Substitutes,Bari, Italy: Jun. 19-20, 1987, Proceedings pp. 135-166.

There is a need for perfluorocarbon emulsions that exhibit both storagestability in the nonfrozen state and a rapid rate of elimination fromthe body. Accordingly, it is an object of the invention to providefluorocarbon emulsions having these characteristics.

SUMMARY OF THE INVENTION

The present invention involves stabilization of fluorocarbon emulsionswith higher molecular weight fluorocarbons that include a lipophilicmoiety. Alternatively, any fluorocarbon having a critical solutiontemperature that is 10EC or more below that which is predicted by itsmolecular weight can be used to stabilize fluorocarbon emulsions inaccordance with this invention.

A major advantage of the present invention is the surprisingly shortorgan retention times of the stabilized emulsion. Perfluorodecylbromide, for example, has a calculated half life in vivo in organs ofthe reticuloendothelial system (RES) of approximately 18 days, whilethose of nonlipophilic perfluorocarbons having about the same molecularweight vary from about 50 to 300 days. (See Table IV.) This distinctionis critical; it spells the difference between formulations which arephysiologically acceptable and those which are not. Note that none ofthe prior art stabilizers are lipophilic; thus, none share theadvantageous properties of the present invention. For example, withreference to Table [V and FIG. 5, the stabilizers of the presentinvention all have critical solution temperatures (CSTs) and projectedorgan retention times much lower than those of the prior art stabilizersof Davis, et al., Kabalnov, and Meinert. Aside from the stabilizers ofthe present invention, conventional fluorocarbons exhibit a directcorrelation between retention time in RES organs and molecular weight.Also, aside from the lipophilic fluorocarbons used in the presentinvention, the perfluorochemical structure has little effect on thestrong retention time/molecular weight relationship. Thus, the presenceof heteroatoms or cyclic structure has little effect on organ retentiontime.

Another major advantage of the present invention over the prior art isthat the emulsions are remarkably stable. This is particularly true whenboth the major (first) fluorocarbon and the stabilizing (second)fluorocarbon include lipophilic moieties.

Thus, in accordance with one aspect of the present invention, there isprovided a storage stable fluorocarbon emulsion, comprising a continuousaqueous phase, an effective amount of an emulsifying agent, and adiscontinuous fluorocarbon phase, comprising from about 50% to about99.9% of a one or more first fluorocarbons, and from about 0.1% to about50% of one or more second fluorocarbons having a molecular weightgreater than each such first fluorocarbon, wherein each such secondfluorocarbon includes at least one lipophilic moiety. The firstfluorocarbon can be selected from a variety of materials, including bis(F-alkyl) ethenes, perfluoroethers having the general structureC_(n)F_(2n+1) —O—C_(n)—F_(2n+1), wherein the sum of n and n′ equals 6 to8, perfluoromethylbicyclo [3.3.1]-nonane,perfluoro-2,2,4,4-tetramethylpentane, perfluorotripropylamine,bis(F-butyl)ethene, (F-isopropyl) (F-hexyl) ethene,perfluoromethyladamantane, perfluorodimethyladamantane,F—N-methyldecahydroisoquinoline, F-4-methyloctahydroquinolidizine,perfluorodecalin, or most preferably, perfluorooctyl bromide. In oneembodiment, each first fluorocarbon has a molecular weight from about460 Daltons to about 550 Daltons, and also preferably has a half life invivo of less than about 4 weeks, preferably less than 2 or 3 weeks, andmost preferably 7 days or less. In the second fluorocarbon, thelipophilic moiety or moities are advantageously Br, Cl, I, H, CH₃, or asaturated or unsaturated hydrocarbon chain of 2 or 3 carbon atoms. Inone preferred embodiment, the second fluorocarbon is an aliphaticperfluorocarbon having the general formula C_(n)F_(2n+1)R orC_(n)F_(2n)R₂, wherein n is an integer from 9 to 12 and R is thelipophilic moiety. In various preferred embodiments, the secondfluorocarbon is selected from the group consisting of perfluorododecylbromide, C₁₀F₂₁CH═CH₂, or C₁₀F₂]CH₂CH₃, or linear or branched brominatedperfluorinated alkyl ethers. Most preferably, the second fluorocarboncomprises perfluorodecyl bromide. It is desirable that each secondfluorocarbon has a molecular weight greater than about 550 Daltons.Pursuant to an alternative definition of the second fluorocarbon, eachsecond fluorocarbon has a critical solution temperature in hexane atleast 10EC lower than that of a fully fluorinated fluorocarbon havingsubstantially the same molecular weight (i.e., a molecular weight within10, and preferably within 3, 4, or 5 daltons). In preferred emulsions,the discontinuous fluorocarbon phase comprises from about 60% to about99.5% of the first fluorocarbon, and from about 0.5% to about 40% of thesecond fluorocarbon; more preferably from about 80% to about 99% of thefirst fluorocarbon, and from about 1% to about 20% of the secondfluorocarbon. A particularly preferred emulsifier is egg yolkphospholipid, and preferred amounts of this emulsifier are 1%-10% w/v.Also preferred are the fluorinated surfactants.

Another aspect of the present invention comprises a method for impartingparticle size stability to a fluorocarbon emulsion having adiscontinuous phase of one or more first fluorocarbons and a continuousaqueous phase, comprising the step of including in admixture with saidfirst fluorocarbon an emulsion-stabilizing amount of one or more secondfluorocarbons having a molecular weight greater than said firstfluorocarbon, wherein each said second fluorocarbon includes within itsstructure a lipophilic moiety. In this method, the definitions of thefirst and second fluorocarbons, the surfactant, and the various emulsionparameters can be the same as for the emulsions discussed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents accelerated stability testing (T=40EC) for 90% w/vfluorocarbon, 4% w/v egg yolk phospholipid emulsions containing mixturesof perfluorooctyl bromide and perfluorodecyl bromide. The stability ofemulsions with 0%, 1%, and 10% w/w perfluorodecyl bromide are presentedin plots of diameter cubed (μm³) vs. time (months).

FIG. 2 (a-d) represents represents particle size histograms (as obtainedby photosedimentation) after three months storage at 40 EC for a 90% w/vfluorocarbon emulsion containing 95/5% w/w mixture of perfluorooctylbromide to perfluorodecyl bromide prepared under similar conditions tothose of FIG. 1. The emulsions are stabilized by 4% w/v egg yolkphospholipid. (Note the emulsion particle diameters as reported on theFigures are not corrected for the vesicle fraction which shows up as apeak in the first histogram bar).

FIG. 3 represents accelerated stability testing (T=40EC) for 60% w/vfluorocarbon, 4% w/v egg yolk phospholipid emulsions containing mixturesof perfluorooctyl bromide and perfluorodecyl bromide. The stability ofemulsions with 0% and 10% w/w perfluorodecyl bromide are presented inplots of diameter cubed (μm³) vs. time (months).

FIG. 4 (a,b) represents a plot of percent mouse lethality vs. dose(ml/kg) for a 3% egg yolk phospholipid, 90% w/v fluorocarbon emulsioncontaining 90%/10% w/w perfluorooctyl bromide/perfluorodecyl bromide.The LD₅₀ of this emulsion is approximately 48 ml/kg.

FIG. 5 represents a plot of fluorocarbon molecular weight (g/mol) versuscritical solution temperature against hexane (EK) for variousfluorocarbons including the prior art emulsion stabilizers proposed byDavis, Meinert, and Kabalnov.

FIG. 6 is a plot of the organ half-life in days vs. molecular weight ofthe fluorocarbon in g/mol. Traditionally, the molecular weight regionfrom 460-550 g/mol has been considered optimal for blood substituteapplications. The lower cutoff is related to the formation of gas embolifor fluorocarbons with vapor pressures greater than 20 torr. The uppercutoff is limited to compounds with organ retention times of less than 3weeks. It is clear that the lipophilic compounds do not fit the generaltrend in that they have shorter organ retention times than would bepredicted for their molecular weight. PFDB has a half-life less than theprescribed 3 week cutoff. (Note—because the organ half-life depends ondose and method of measurement, the values for PFOB and PFDB have beenscaled relative to F-decalin knowing that the ratio of half-lives forPFOB/FDC=4/7).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Introduction

The fluorocarbon emulsions of the present invention comprise two phases:a continuous aqueous phase and a discontinuous fluorocarbon phase.Osmotic agents and buffers, generally, are also included in thecontinuous phase to maintain osmolarity and pH to promote physiologicalacceptability.

The discontinuous phase of modern fluorocarbon emulsions for therapeuticuse generally comprises from 20% w/v to up to 125% w/v of a fluorocarbonor a highly fluorinated compound (hereinafter referred as a“fluorocarbon” or a “perfluorocarbon”). As used herein, the expression“weight per volume” or “w/v” will mean grams per 100 cubic centimetersor milliliters. Also, as used herein, the expression “weight per weight”or “w/w” will be used and understood to mean the weight fractions ofcomponents that add up to give a desired weight per volume.

The present invention provides stable fluorocarbon emulsions by formingthe discontinuous phase from a mixture of at least two fluorocarbons, atleast one of which has a relatively higher molecular weight and whichincludes in its molecular structure a lipophilic moiety. Unlike priorart emulsions in which a higher molecular weight fluorocarbon isincluded to prevent Ostwald ripening, the present added fluorocarbon(s)are excreted at a rate which is physiologically acceptable. Stablefluorocarbon emulsions with particle sizes as small as ca. 0.1 μm may beprepared, with good particle size stability. Surprisingly, emulsions ofthe present invention may be stored with little or no particle growth atrelatively elevated temperatures of 20EC to 40EC.

A preferred embodiment utilizes two fluorocarbons; however, the “first”and “second” fluorocarbons discussed below can instead comprise mixturesof fluorocarbons, each of which has the specified characteristics.

The first fluorocarbon preferably has a molecular weight from about 460to 550 Daltons and is employed in a relative ratio of 50% to 99.9% byweight. The second fluorocarbon is preferably an aliphatic fluorocarbon,including within its molecular structure at least one lipophilic moiety,and having a molecular weight greater than about 550 Daltons at arelative ratio of 50% to 0.1%. Linear fluorocarbons are preferred forboth the first and second fluorocarbons. The second fluorocarbon ispreferably terminally substituted with the lipophilic moiety, althoughsubstitutions at other positions are also contemplated.

A first alternative definition of the second fluorocarbon focuses on itscritical solution temperature (CST). In accordance with this definition,the second fluorocarbon has a CST that is lower than the CST of a fullyfluorinated fluorocarbon (lacking a lipophilic moiety) havingsubstantially the same molecular weight. Preferably, the CST of thesecond fluorocarbon is at least 10E lower than such a fully fluorinatedfluorocarbon.

A second alternative definition of the second fluorocarbon focuses onits organ retention time. It is possible to predict organ retention timefrom a log plot of the molecular weight of the fluorocarbon. In thepresent invention, the second fluorocarbon preferably has an organretention time that is less than what is predicted by the aforementionedlog plot.

Emulsions may be prepared through the method of the present invention atvery high fluorocarbon concentrations (up to 125%, w/v), virtually anydesired particle size, and with very low quantities of emulsifyingagents, without losing stability. Unlike prior art, stabilizedfluorocarbon emulsions, the organ retention time of the addedfluorocarbon is well within acceptable limits. Further advantages andattributes are discussed below.

The Compositions

A. The Discontinuous Phase

The characteristics of fluorocarbons suitable for use in the presentinvention are discussed in more detail below. Examples of suitablefluorocarbons are provided.

The First Fluorocarbon

The first fluorocarbon is selected for its short organ retention timeand biocompatibility. In general, the half life in organs is preferablyless than about 4 weeks, more preferably less than about 2 or 3 weeks,and most preferably 7 days or less. The molecular weight is from about460 to about 550 daltons.

Such fluorocarbons include bis(F-alkyl)ethenes such as C₄F₉CH═CHC₄F₉(“F-44E”), i-CF₃CF₉CH═CHC₆F₁₃ (“F-i36E”), and cyclic fluorocarbons, suchas C₁₀F₁₈ (F-decalin, perfluorodecalin or FDC); F-adamantane (FA);perfluoroindane; F-methyladamantane (FMA); F-1,3-dimethyladamantane(FDMA); perfluoro-2,2,4,4-tetramethylpentane; F-di- orF-tri-methylbicyclo[3,3,1]nonane (nonane); C₇₋₁₂ perfluorinated amines,such as F-tripropylamine, F-4-methyloctahydroquinolizine (FMOQ),F-n-methyl-decahydroisoquinoline (FMIQ), F-n-methyldecahydroquinoline(FHQ), F-n-cyclohexylpyrrolidine (FCHP), and F-2-butyltetrahydrofuran(FC-75 or RM101).

Other examples of appropriate first fluorocarbons include brominatedperfluorocarbons, such as perfluorooctyl bromide (C₈F₁₇Br, USANperflubron), 1-bromopentadecafluoroheptane (C₇ F₁₅Br), and1-bromotridecafluorohexane (C₆ F₁₃Br, also known as perfluorohexylbromide or PFHB. Other brominated fluorocarbons are disclosed in U.S.Pat. Nos. 3,975,512 and 4,987,154 to Long.

Also contemplated are fluorocarbons having other nonfluorinesubstituents, such as 1-chloro-heptadecafluorooctane (C₈F₁₇Cl, alsoreferred to as perfluorooctyl chloride or PFOCl); perfluorooctylhydride, and similar compounds having different numbers of carbon atoms.

Additional first fluorocarbons contemplated in accordance with thisinvention include perfluoroalkylated ethers, halogenated ethers(especially brominated ethers), or polyethers, such as(CF₃)₂CFO(CF₂CF₂)₂OCF(CF₃)₂; (C₄F₉)₂O. Further, fluorocarbon-hydrocarboncompounds may be used, such as, for example compounds having the generalformula C_(n)F_(2n+1) —C_(n′)H_(2n′+1); C_(n)F_(2n+1) OC_(n′)H_(2n′+1);or C_(n)F_(2n+1)CH═CHC_(n′)H_(2n′+1), wherein n and n′ are the same ordifferent and are from about 1 to about 10 (so long as the compound is aliquid at room temperature). Such compounds, for example, includeC₈F₁₇C₂H₅ and C₆ F₁₃CH═CHC₆H₁₃.

Particularly preferred fluorocarbons for use as the first fluorocarboninclude perfluoroamines, terminally substituted linear aliphaticperfluorocarbons having the general structure:

-   -   C_(n)F_(2n+1)R, wherein n is an integer from 6 to 8 and R        comprises a lipophilic moiety selected from the group of Br, Cl,        I, CH₃, or a saturated or unsaturated hydrocarbon of 2 or 3        carbon atoms,        bis (F-alkyl) ethenes having the general structure:    -   C_(n)F_(2n+1)—CH═CH—C_(n′)F_(2n′+1), wherein the sum of n and n′        equals 6 to 10, and        perfluoroethers having the general structure:    -   C_(n)F_(2n+1)—O—C_(n′)F_(2n′+1), wherein the sum of n and n′        equals 6 to 9.

In addition, fluorocarbons selected from the general groups ofperfluorocycloalkanes or perfluoroalkyl-cycloalkanes, perfluoroalkylsaturated heterocyclic compounds, or perfluorotertiary amines may besuitably utilized as the first fluorocarbon. See generally Schweighart,U.S. Pat. No. 4,866,096.

It will be appreciated that esters, thioethers, and other variouslymodified mixed fluorocarbon-hydrocarbon compounds, including isomers,are also encompassed within the broad definition of fluorocarbonmaterials suitable for use as the first fluorocarbon of the presentinvention. Other suitable mixtures of fluorocarbons are alsocontemplated.

Additional fluorocarbons not listed here, but having the propertiesdescribed in this disclosure that would lend themselves to therapeuticapplications, are also contemplated. Such fluorocarbons may becommercially available or specially prepared. As will be appreciated byone skilled in the art, there exist a variety of methods for thepreparation of fluorocarbons that are well known in the art. See forexample, Schweighart, U.S. Pat. No. 4,895,876.

The Second Fluorocarbon

The second fluorocarbon is an aliphatic fluorocarbon substituted withone or more lipophilic moieties and having a higher molecular weightthan the first fluorocarbon. Advantageously, the lipophilic moiety is aterminal substitution on the fluorocarbon molecule. Preferably, themolecular weight of the second fluorocarbon is greater than about 540Daltons. Constraints on the upper limit of the molecular weight of thesecond fluorocarbon will generally be related to its organ retentiontime and its ability to be solubilized by the first fluorocarbon.Usually, the second fluorocarbon has a molecular weight less than about700 Daltons.

Most preferred second fluorocarbons have boiling points greater thanabout 150EC and water solubilities of less than about 1×10⁻⁹moles/liter.

Of course, as will be appreciated by one skilled in the art, manyfluorocarbons substituted with different lipophilic groups could besuitably used as the second fluorocarbon in the present invention. Suchfluorocarbons may include esters, thioethers, and variousfluorocarbon-hydrocarbon compounds, including isomers. Mixtures of twoor more fluorocarbons satisfying the criteria set forth herein are alsoencompassed within the broad definition of fluorocarbon materialssuitable for use as the second fluorocarbon of the present invention.Fluorocarbons not listed here, but having the properties described inthis disclosure that would lend themselves to therapeutic applicationsare additionally contemplated.

The lipophilic moiety is optimally selected from the group consisting ofBr, Cl, I, CH₃, or a saturated or unsaturated hydrocarbon of 2 or 3carbon atoms. Consequently, preferred second fluorocarbons may beselected from the group of terminally substituted perfluorocarbonhalides as represented by the general formula:

-   -   C_(n)F_(2n+1)X or C_(n)F_(2n)X₂, wherein n is 8 or greater,        preferably 10 to 12, and X is a halide selected from the group        consisting of Br, Cl, or I;        1-alkyl-perfluorocarbons or dialkylperfluorocarbons as        represented by the general formula:    -   C_(n)F_(2n+1)—(CH₂)_(n′)CH₃ wherein n is 8 or greater,        preferably 10 to 12, and n′ is 0 to 2;        1-alkenyl-perfluorocarbons as represented by the general        formula:    -   C_(n)F_(2n+1)—C_(n′)H_((2n′−1), wherein n is 10 or more,        preferably 10 to 12, and n′ is either 2 or 3; or        brominated linear or branched perfluoroethers or polyethers        having the following general structure:    -   Br—(C_(n)F_(2n+1)—O—C_(n′)F_(2n′+1)), wherein n and n′ are each        at least 2 and the sum of n and n′ is greater than or equal to        8.

Most preferably, the second fluorocarbon of the present invention isselected from the group consisting of linear or branched brominatedperfluorinated alkyl ethers, perfluorodecyl bromide (C₁₀F₂₁Br);perfluorododecyl bromide (C₁₂F₂₅Br); 1-perfluorodecylethane(C₁₀F₂₁CH═CH₂); and 1-perfluorodecylethene (C₁₀F₂₁CH₂CH₃); withperfluorodecyl bromide particularly preferred.

In accordance with a first alternative definition, whether or not theysatisfy the foregoing definitions, fluorocarbons having criticalsolution temperatures (CSTs) vs hexane more than 10EC below the CST of afluorocarbon having substantially the same molecular weight (variationsof up to about 10 daltons being acceptable) are also suitable for use inthe present invention. A comparison between the CST and molecular weightof a number of perfluorocarbons is presented in Table IV, below.Methodology for determining CST is presented in Example 9.

A second alternative definition of the second fluorocarbon is evidentfrom FIG. 6 and Example 8. Suitable second fluorocarbons may be selectedfrom those which have significantly lower half lives than nonlipophilicfluorocarbons of substantially the same molecular weight. As isevidenced in FIG. 6, the log of the half life in days of suitable secondfluorocarbons can be at least 0.2, preferably at least 0.3, and morepreferably at least 0.4 or 0.5 less than the average or expected valuefor nonlipophilic fluorocarbons of substantially the same molecularweight.

The Emulsifying Agent

The fluorocarbon emulsions also include an emulsifying agent. As used inthis specification, an emulsifying agent is any compound or compositionthat aids in the formation and maintenance of the droplets of thediscontinuous phase by forming a layer at the interface between thediscontinuous and continuous phases. The emulsifying agent may comprisea single compound or any combination of compounds, such as in the caseof co-surfactants.

In the present invention, preferred emulsifying agents are selected fromthe group consisting of phospholipids, nonionic surfactants, fluorinatedsurfactants, which can be neutral or anionic, and combinations of suchemulsifying agents.

Lecithin is a phospholipid that has frequently been used as afluorocarbon emulsifying agent, as is more fully described in U.S. Pat.No. 4,865,836. Egg yolk phospholipids have shown great promise asemulsifying agents for fluorocarbons. See e.g., Long, U.S. Pat. No.4,987,154.

Other emulsifying agents may be used with good effect, such asfluorinated surfactants, also known as fluorosurfactants.Fluorosurfactants that can provide stable emulsions includetriperfluoroalkylcholate; perfluoroalkylcholestanol;perfluoroalkyloxymethylcholate; C₃F₇O(CF₂)₃C(═O)NH(CH₂)₃N(O)(CH₃)₂(XMO-10); and fluorinated polyhydroxylated surfactants, such as, forexample, those discussed in “Design, Synthesis and Evaluation ofFluorocarbons and Surfactants for In Vivo Applications NewPerfluoroalkylated Polyhydroxylated Surfactants” by J. G. Riess, et al.J. G. Riess et al.; Biomat. Artif. Cells Artif. Organs 16: 421-430(1988).

The nonionic surfactants suitable for use in the present inventioninclude polyoxyethylene-polyoxypropylene copolymers. An example of suchclass of compounds is Pluronic, such as Pluronic F-68. Anionicsurfactants, particularly fatty acids (or their salts) having 12 to 24carbon atoms, may also be used. One example of a suitable anionicsurfactant is oleic acid, or its salt, sodium oleate.

It will be appreciated that choice of a particular emulsifying agent isnot central to the present invention. Indeed, virtually any emulsifyingagent (including those still to be developed) capable of facilitatingformation of a fluorocarbon-in-water emulsion can form improvedemulsions when used in the present invention. The optimum emulsifyingagent or combination of emulsifying agents for a given application maybe determined through empirical studies that do not require undueexperimentation. Consequently, one practicing the art of the presentinvention should choose the emulsifying agent or combination ofemulsifying agents for such properties as biocompatibility.

B. The Continuous Phase

The continuous phase comprises an aqueous medium. Preferably, the mediumis physiologically acceptable. For instance, a preferred emulsion willhave the ability to buffer and maintain pH, as well as provide anappropriate osmolarity. This typically has been achieved in the artthrough the inclusion in the aqueous phase of one or more conventionalbuffering and/or osmotic agents, or an agent that combines theseproperties.

Additionally, one may supplement the continuous phase with other agentsor adjuvants for stabilizing or otherwise increasing the beneficialaspects of the emulsion. These agents or adjuvants include: steroidhormones, cholesterol, tocopherols, and/or mixtures or combinationsthereof. Suitable steroid hormones include fluorinated corticosteroids.

C. Preparation of the Emulsion

Fluorocarbon emulsions according to the invention are prepared by meansof conventional emulsification procedures, such as, for example,mechanical or ultrasonic emulsification of an emulsion formulation in aManton-Gaulin mixer or Microfluidizer (Microfluidics Corp., Newton,Mass.) as described in Example 1.

The first and second fluorocarbons are combined with the aqueous phasein the desired ratios, together with the surfactant. Usually, apreemulsion mixture is prepared by simple mixing or blending of thevarious components. This preemulsion is then emulsified in the desiredemulsification apparatus.

The second fluorocarbon can comprise from about 0.1% to 50% (w/w) of thetotal amount of fluorocarbon; in preferred embodiments, the secondfluorocarbon comprises from about 0.5% to about 40% of the total amountof fluorocarbon, with the first fluorocarbon comprising the remainder ofthe total fluorocarbon. The combined fluorocarbon concentration in theemulsion is preferably anywhere within the range of about 20% to about125% (w/v). In preferred emulsions, the total perfluorocarbonconcentration is from about 30%, 40%, or 50% to about 70%, 80%, 90%, or100% (w/v). Emulsifiers are added in concentrations of from about 0.1%to 10%, more preferably 1% or 2% to about 6% (w/v).

Effect of Stabilizer on Emulsion Particle Size

The addition of stabilizing second fluorocarbons such as perfluorodecylbromide to 90% perfluorocarbon emulsion comprising perfluorooctylbromide provides substantial decreases in the range of particle size andimproves particle size stability (Example 3, Table 1). The data in FIG.1 representing average particle growth size in 90% w/v perfluorocarbonemulsions containing 1% and 10% perfluorodecyl bromide (PFDB) indicatethat the stabilizing effect of PFDB on particle size can be observed inemulsion comprising only 1% w/w PFDB, and that substantial improvementoccurs at the 10% w/w concentration level. The data of FIG. 2 indicatethat PFDB at an intermediate concentration of 4.5% w/v (equivalent to 5%w/w of total perfluorocarbon) also maintains a narrower distribution ofparticle sizes in a 90% w/v perfluorocarbon emulsion after 3 months ofaging as compared to a non-stabilized emulsion. Perflubron emulsionsstabilized with PFDB also have smaller initial particle sizes (Tables Iand II, FIG. 2).

The stabilizing effect of PFDB in perfluorocarbon emulsion is alsoindependent of total fluorocarbon concentration (Table II, FIG. 3).Emulsions comprising 60% w/v or 90% w/v perfluorocarbon, consisting of10% w/w PFDB and 90% w/w perflubron, demonstrated similar initialaverage particle sizes and particle size stability on aging. Thestabilizing effect of PFDB also operates in perfluorodecalin emulsions(Example 6, Table III), to produce emulsions having a small initialparticle size and to substantially retain that size during three monthsof aging at 40 EC.

The stabilizing effect of PFDB operates in both perflubron andperfluorodecalin emulsions (Example 7) comprising either 60% or 90%(Table IV) to produce emulsions having a small initial average particlesize and to retain that size and substantially retaining that sizeduring 3 months of aging at 40EC.

Further details of the method of the present invention can be morecompletely understood by reference to the following illustrativeExamples.

EXAMPLE 1

Preparation of Reference Emulsion

Composition of Reference Emulsion:

Perflubron/Lecithin (90/4% w/v)

A reference emulsion containing 90 g PFOB, 4 g egg yolk phospholipid(EYP), and physiological levels of salts and buffers was prepared byhigh pressure homogenization according to the method of Long (U.S. Pat.No. 4,987,154).

EXAMPLE 2

Stabilization of a 90% w/v Fluorocarbon Emulsion (PerfluorooctylBromide/Perfluorodecyl Bromide)

The protocol of Example 1 was repeated to form four additionalemulsions, except that in successive emulsions, the fluorocarbon wasperfluorooctyl bromide containing 1%, 2%, 5%, and 10% perfluorodecylbromide (w/w), respectively.

EXAMPLE 3

Emulsion Stability

The emulsions prepared by the procedures of Examples 1 and 2 were placedon accelerated stability testing at 40EC for three months. Table Idemonstrates particle size stability over time for 90% (w/v)fluorocarbon emulsions. Such emulsions include a control, in which 100%of the fluorocarbon phase is perfluorooctyl bromide, and emulsions ofthe present invention in which the fluorocarbon phase is 99% to 90% w/wperfluorooctyl bromide, with from 1% to 10% w/w of perfluorodecylbromide added as a stabilizer. In FIG. 1 and Table I, “EYP” is egg yolkphospholipid, “perflubron” is perfluorooctyl bromide, “PFDB” isperfluorodecyl bromide, and “S” is the rate of particle growth in unitsof μm³/mo. FIG. 1 illustrates typical Lifshitz-Slezov graphs of d³ as afunction of time for these emulsions. The cubed term is chosen for theordinate since Lifshits-Slezov theory predicts that plots of d³ vs timewill yield a straight line. In fact, this linear dependence is generallyobserved for fluorocarbon emulsions. TABLE I Stabilizing Effect ofPerfluorodecyl bromide (90% w/v Emulsion ContainingPerflubron/Perfluorodecyl bromide (PFDB) with 4% EYP, T = 40EC) SizeSize Size Initial After After After PFDB Size One Two Three % w/w (μm)Month Months Months S × 1000  0% 0.23 0.39 0.49 0.52 44.4 (0.13) (0.19)(0.20) (0.23)  1% 0.23 0.29 0.37 0.37 14.2 (0.12) (0.16) (0.19) (0.18) 2% 0.19 0.23 0.26 0.32 8.3 (0.09) (0.12) (0.14) (0.17)  5% 0.18 0.200.24 0.28 5.4 (0.08) (0.10) (0.13) (0.14) 10% 0.20 0.25 0.27 0.27 3.9(0.12) (0.13) (0.14) (0.16)(parentheses indicate the standard deviation in the distribution ofparticle sizes)

EXAMPLE 4

Stabilization of a 60% w/v Fluorocarbon Emulsion (PerfluoroctylBromide/Perfluorodecyl Bromide)

Table II compares particle size increase in a 60% w/v perflubronemulsion containing perfluorodecyl bromide with a particle size increasein a reference emulsion that does not contain PFDB. TABLE IIStabilization of a 60% w/v Fluorocarbon Emulsion (PerfluoroctylBromide/Perfluorodecyl Bromide) Size Initial after 1 Size after 2 Sizeafter 3 Size month months months S × 1000 Sample (μm) (40EC) (40EC)(40EC) (μm³/mo)  0% w/v 0.20 0.34 0.38 0.39 16.9 PFDB 10% w/v 0.18 0.200.23 0.23 2.3 PFDB

EXAMPLE 5

In Vivo Data

FIG. 4 graphs the % lethality in mice injected with various doses of a90% w/v fluorocarbon containing 90% w/w perfluorooctyl bromide and 10%w/w perfluorodecyl bromide, along with 3% w/v EYP. The LD50 wasapproximately 48 ml/kg. FIG. 9 graphs the LD₅₀ in mice of a 90% w/vperfluorocarbon emulsion of the present invention, consisting of 90% w/wPerflubron, as the first fluorocarbon, and 10% perfluorodecyl bromide,as the second fluorocarbon, emulsified with 3% w/v egg yolkphospholipid. The LD₅₀ was approximately 48 ml/kg.

EXAMPLE 6

Stability of Perfluorodecalin/Perfluorodecyl bromide Emulsions

Concentrated perfluorodecalin emulsions were prepared according to theprocedure of Examples 1 and 2 are studied for stability as described inExample 3. Stability data is presented in Table III. TABLE IIIStabilizing Effect of Perfluorodecyl Bromide on ConcentratedPerfluorodecalin Emulsions Size Size Size Initial after 1 after 2 after3 Size month months months S × 1000 Sample (μm) (40EC) (40EC) (40EC)(μm³/mo) 58.2% w/v FDC 0.17 0.33 0.40 0.49 36.6  4.6% w/v EYP 58.2% w/vFDC 0.18 0.19 0.22 0.25 3.3   10% w/v PFDB  4.6% EYP   81% w/v FDC 0.240.34 0.45 0.57 56.6   3% w/v EYP   81% w/v FDC 0.19 0.22 0.25 0.27 4.3  9% w/v PFDB   3% EYP

EXAMPLE 7

Predicted Organ Retention Times

Table IV compares physical data (including organ retention times ofstabilizing fluorocarbons suggested in the prior art with comparabledata for compounds of the present invention. Note that, although themolecular weight and boiling point of F-decylbromide are comparable tothe prior art, the critical solution temperature (which is related toorgan retention time) is dramatically lower.

FIG. 5 illustrates the critical solution temperatures of severalfluorocarbons as a function of molecular weight. Note that the prior artstabilizers of Davis, et al., Meinert, Perftoran, and Green Cross(F-tripropylamine), and 50 other assorted fluorocarbons all exhibit apredictable relationship between CST and molecular weight.

On the other hand, the lipophilic stabilizers of the present inventionhave substantially lower CSTs, and therefore substantially shorter organretention half lives. TABLE IV Physical Properties of Minor ComponentsDiscussed in Literature (Proposed Minor Components are listed inBoldface) Name Formula (days) t_(1/2) MW(g/mol) b.p.(C) CSTH(C) Davis,et al. (U.S. Pat. No. 4,859,363) F-perhydrofluorene C₁₃F₂₂ 574 192-193n.a. F-perhydrophenanthrene C₁₄F₂₄ 624 215-216 48 F-perhydrofluorantheneC₁₆F₂₆ 686 242-243 n .a. Kabalnov, et al. (Kolloidin Zh. 4827-32(1986))F-N-methylcyclohexylpiperidine C₁₂F₂₁N 557 n.a. 40 Meinert (U.S. Pat.No. 5,120,731) F-N-cyclohexylmorpholine C₁₀F₁₈NO 492 n.a. 31F-dimorpholinoethane C₁₀F₂₀N₂O₂ 560 164 38 F-dimorpholinopropaneC₁₁F₂₂N₂O₂ 610 182 45 F-dimorpholinopentane C₁₃F₂₆N₂O₂ 710 215 60F-dipiperidine C₁₀F₁₆N₂ 452 145-150 36 F-dipiperidinomethane C₁₁F₁₈N₂502 165-175 42 F-dipiperidinoethane C₁₂F₂₀N₂ 552 181-186 49F-dipiperidinopropane C₁₃F₂₂N₂ 602 195-203 56 F-dipiperidinobutaneC₁₄F₂₄N₂ 652 231-238 72 Present Study F-decalin C₁₀F₁₈ 462 142 22F-hexyl bromide C₆F₁₃Br 399 n.a. n.a. F-octyl bromide C₈F₁₇Br 499 143(−19)^(a) F-decyl bromide C₁₀F₂₁Br 599 (198)^(b) (19)^(a)F-bromopolyether C₁₁F₂₃O₃Br 697 n.a. 32^(a)values for the critical solution temperature with hexane areestimated from extrapolations linear plots of the critical solutiontemperature vs. hydrocarbon chain length.^(b)the value of the boiling point of F-decyl bromide is estimated fromHildebrand solution theory.

EXAMPLE 8

Organ Retention Time vs. Fluorocarbon Molecular Weight

FIG. 6 presents data for organ retention times vs. molecular weight fora number of fluorocarbons. The fluorocarbons of Meinert, Kabalnov, andDavis are all contained within the large group, which show a tightcorrelation between organ retention time and fluorocarbon molecularweight. Based on this the optimal molecular weight for blood substituteapplications has been defined to be 460-550 g/mol. The lower limit isdefined by fluorocarbons with vapor pressures greater than 20 torr whichcauses gas emboli, while the upper limit is defined by fluorocarbonswith organ retention times of less than 3 weeks. It is clear from FIG. 6that the lipophilic fluorocarbons PFOB, PFDB, and PPEB(perfluoropolyether bromide, C₁₁F₂₃O₃Br) have organ retention times lessthan be predicted from the molecular weight. Due to their decreasedwater solubilities (which follows with molecular weight) PFDB and PPEBare expected to stabilize fluorocarbon emulsions by decreasing Ostwaldripening.

EXAMPLE 9

Measurement of Critical Solution Temperature (CST)

Critical solution temperature for fluorocarbon liquids was measured inthe following manner: Equivolume mixtures of the test fluorocarbon andhydrocarbon (e.g., hexane) are placed in a sealed vial and submerged ina temperature controlled water bath. Samples are cooled until twodistinct phases are present. At this point, the temperature is increasedslowly. The lowest temperature at which the two phases are completelymiscible (i.e., a single liquid phase) is defined as the CST.

For comparison purposes, all CST temperatures used in this patent arereported versus hexane. It is often not possible, however, to measurethe CST for lipophilic fluorocarbons versus hexane, since the CSTs forthese substances are very low. Thus, the CST for lipophilic substancesis often measured in longer chain length hydrocarbons, and the valueversus hexane is determined via extrapolation of linear plots of CST vs.alkane chain length.

Although the present invention has been disclosed in the context ofcertain preferred embodiments, it is intended that the scope of theinvention be measured by the claims that follow, and not be limited tothose preferred embodiments:

1. A stabilized fluorocarbon emulsion comprised of a continuous anddiscontinuous phase for use in in vivo oxygen transport, comprising: amixture of fluorocarbons comprising at least a first fluorocarbon fromabout 40-60% w/v of the emulsion and a second fluorocarbon from about1-10% w/v of the emulsion; at least one emulsifying agent selected fromthe group consisting of lecithins, fluorinated surfactants and salts;wherein the first and second fluorocarbons make up the discontinuousphase of the emulsion and the first fluorocarbon is comprised of afluorocarbon having a molecular weight greater than 460 daltons and asecond fluorocarbon having a molecular weight greater than about 540daltons, wherein the first and second fluorocarbon each have at leastone lipophilic moiety selected from the group consisting of Br, Cl andI; and the continuous phase is comprised of a pharmaceuticallyacceptable aqueous carrier.
 2. The stabilized fluorocarbon emulsion ofclaim 1 wherein the stabilized emulsion is effective in transportingoxygen to the tissues throughout the body when the emulsion is saturatedwith oxygen and injected in vivo.
 3. The stabilized fluorocarbonemulsion of claim 1 wherein the first fluorocarbon is selected from thegroup consisting of bis(F-alkyl) amines, cyclic fluorocarbons, C₇₋₁₂perfluorinated amines, brominated fluorocarbons, perfluoroalkylatedethers, halogenated ethers and poly ethers all having a molecular weightgreater than 460 daltons.
 4. The stabilized fluorocarbon emulsion ofclaim 1 wherein the first fluorocarbon is a compound of the followinggeneral structure: C_(n)F_(2n+1)R, wherein n is an integer from 6 to 8and R comprises a lipophilic moiety selected from the group consistingof Br, Cl, I, CH₃ or a saturated or unsaturated hydrocarbon of 2 or 3atoms.
 5. The stabilized flourocarbon emulsion of claim 1 wherein thefirst fluorocarbon is perfluorooctyl bromide.
 6. The stabilizedfluorocarbon emulsion of claim 1 wherein the second fluorocarbon is acompound selected from the group of the following compounds: a)C_(n)F_(2n+1)X, wherein n is 8 or greater, preferably 10 to 12, and X isa halide selected from the group consisting of Br, Cl or I; b)C_(n)F_(2n)X₂, wherein n is 8 or greater, preferably 10 to 12, and X₂ isa halide selected from the group consisting of Br, CL or I; and, c)Br—(C_(n)F_(2n+1)—O—C_(n′)F_(2n′+1)) wherein n and n′ are each at least2 and the sum of n and n′ is greater or equal to
 8. 7. The stabilizedfluorocarbon emulsion of claim 1 wherein the second fluorocarbon is acompound selected from the group consisting of perfluorodecyl bromide,perfluoropolyether bromide and perfluorododecyl bromide.
 8. Thestabilized fluorocarbon emulsion of claim 1 wherein lecithin is an eggyolk phospholipid.
 9. The stabilized flouorocarbon emulsion of claim 1wherein the emulsion further comprises tocopherol.
 10. The stabilizedfluorocarbon emulsion of claim 1 wherein the emulsion further comprisesNaCl.
 11. An emulsion for use in in vivo oxygen transport consisting ofa continuous and discontinuous phase, wherein the discontinuous phaseconsists of two fluorocarbons, wherein the first fluorocarbon isselected from the group consisting of: a) C_(n)F_(2n+1)X, wherein n is 8or greater, preferably 10 to 12, and X is a halide selected from thegroup consisting of Br, Cl or I; b) C_(n)F_(2n)X₂, wherein n is 8 orgreater, preferably 10 to 12, and X₂ is a halide selected from the groupconsisting of Br, Cl or I; c) Br—(C_(n)F_(2n+1)—O—C_(n′)F_(2n′+1))wherein n and n′ are each at least 2 and the sum of n and n′ is greateror equal to 8; d) C_(n)F_(2n+1)R—CH═CH-C_(n′)F_(2n′+1), wherein the sumof n and n′ equals 6 to 10; and, e) C_(n)F_(2n+1)—O—C_(n′)F_(2n′+1),wherein the sum of n and n′ equals 6 to 10; and the second fluorocarbonis selected from the group consisting of perfluorodecyl bromide,perfluorododecyl bromide, 1-perfluorodecylethylene,1-perfluorodecylethane, the continuous phase being comprised of apharmaceutically acceptable aqueous carrier, and, the emulsion furthercomprising an emulsifying agent.
 12. The emulsion of claim 11 whereinthe emulsifying agent is a phospholipid.
 13. The emulsion of claim 11wherein the emulsifying agent is a surfactant.
 14. The emulsion of claim11 wherein the first fluorocarbon is selected from the group consistingof: C_(n)F_(2n+1)X, wherein n is 8 or greater, preferably 10 to 12, andX is a halide selected from the group consisting of Br, Cl or I; and,C_(n)F_(2n)X₂, wherein n is 8 or greater, preferably 10 to 12, and X₂ isa halide selected from the group consisting of Br, Cl or I.
 15. Theemulsion of claim 11 wherein the second fluorocarbon is perfluorodecylbromide.
 16. The emulsion of claim 11 wherein the first fluorocarbon isperfluorooctyl bromide.
 17. An emulsion for use in in vivo oxygentransport comprising: an aqueous continuous phase, at least oneemulsifying agent comprised of a phospholipid, a discontinuous phase,comprising at least two fluorocarbons wherein the first fluorocarbon hasa molecular weight greater than 460 daltons and the second fluorocarbonhas a molecular weight greater than about 540 daltons, wherein thesecond fluorocarbon has at least one lipophilic moiety selected from thegroup consisting of Br, Cl, I and H; and the continuous phase iscomprised of a pharmaceutically acceptable aqueous carrier.
 18. Theemulsion of claim 17 wherein the first fluorocarbon is selected from thegroup consisting of bis(F-alkyl) amines, cyclic fluorocarbons, C₇₋₁₂perfluorinated amines, brominated fluorocarbons, perfluoroalkylatedethers, halogenated ethers and poly ethers all having a molecular weightgreater than 460 daltons.
 19. The emulsion of claim 17 wherein the firstfluorocarbon is selected from the group consisting of F-44E, F-i36E,perfluorodecalin, F-adamantane, perfluoroindane, F-methyladamantane,FDMA, perfluoro-2,2,4,4-tetramethylpentane,F-di-methylbicyclo[3,3,1]nonane, F-tri-methylbicyclo[3,3,1]nonane,F-tripropylamine, FMOQ, FMIQ, FHQ, FCHP, FC-75, RM11, perfluorooctylbromide, 1-bromotridecafluorohexane, perfluorooctyl chloride andperfluorooctyl hydride.
 20. The emulsion of claim 17 wherein the secondfluorocarbon is selected from the group consisting of compounds havingthe following general structure: a) C_(n)F_(2n+1)X, wherein n is 8 orgreater, preferably 10 to 12, and X is a halide selected from the groupconsisting of Br, Cl or I; b) C_(n)F_(2n)X₂, wherein n is 8 or greater,preferably 10 to 12, and X₂ is a halide selected from the groupconsisting of Br, CL or I; and, c) Br—(C_(n)F_(2n+1)—O—C_(n′)F_(2n′+1))wherein n and n′ are each at least 2 and the sum of n and n′ is greateror equal to
 8. 21. The emulsion of claim 17 wherein the secondfluorocarbon is selected from the group consisting of perfluorodecylbromide, perfluorododecyl bromide, 1-perfluorodecylethylene,1-perfluorodecylethane.